**2.1. Free- ranging cetaceans**

Samples of skin (epidermis, dermis and blubber) were obtained from free-ranging specimens of long-beaked common dolphin (*Delphinus capensis*; MDC12) and common dolphin (*Delphinus delphis*; DDL1) using an aluminium pole armed with biopsy tips (0.7 cm ø, 3.0 cm length), while skin biopsies from free-ranging specimens of Bryde's whale (*Balaenoptera edeni*; MBE3), killer whale (*Orcinus orca*; MOO12), sperm whale (*Physeter macrocephalus*; PMAS1), bottlenose dolphin (*Tursiops truncatus*; TTA1) and Risso's dolphin (*Grampus griseus*; GGL1) were obtained using a Barnett Wildcat II crossbow with a 150 pound test bow, using a biopsy dart with modified stainless steel collecting tip (0.9 cm ø, 4.0 cm length). Biopsy samples were taken in the dorsal area near the dorsal fin, with CITES authorization (CITES Nat. IT025IS, Int. CITES IT 007) in the Sea of Cortez (MDC12, MBE3 and MOO12) and Mediterranean Sea (DDL1, PMAS1, TTA1 and GGL1). A small fragment of the biopsy was immediately stored in cell medium for the cell cultures.

#### **2.2. Stranded cetaceans**

Skin tissue of stranded cetaceans (dead within 2-12 h) were obtained from specimens found dead along the Italian coasts in the period 2005–2009 (CITES Nat. IT025IS, Int. CITES IT 007). Samples were taken under the dorsal fin of stranded specimens of fin whale (RT2 and

RT25), sperm whale (PM6), bottlenose dolphin (TurNic) and striped dolphin (RT1 and RT23), and immediately placed in cell medium.

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

0.01 μg/ml 0.1 μg/ml 1 μg/ml 5 μg/ml <sup>25</sup>

0.01 μg/ml 0.05 μg/ml 0.1 μg/ml / /

0.1 μg/ml 1 μg/ml 10 μg/ml 100 μg/ml /

12.5μM BaP + 250μM BnF

μg/ml

/ /

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 53

**Name Mixture Dose 1 Dose 2 Dose 3 Dose 4 Dose 5** 

2.5μM BaP + 50μM BnF

0.5μM BaP + 10μM BnF

**Table 1.** The different mixtures of contaminants and doses to which cell cultures were exposed.

antibody without cross reaction as the primary antibody was absent.

**5. Genotoxicity biomarker: comet assay** 

Fluor localized in the membrane of cytoplasmatic area of sample cells. The sample cells are imaged using DAPI and this image is presented to the operator for threshold selection of cytoplasmatic and nuclei Region of Interests (ROIs) across the field. The procedure then utilizes these ROIs to measure fluorescence intensity of Alexa Fluor sample cell and summarizes the results in a worksheet. The system generates index values which are unitless until compared with other units, such as number of cells to obtain mean fluorescence per cell or the area in which it is calculated to obtain mean fluorescence per mm2. Images are all obtained with a magnification of 20X, a calibration of 0.65 μm/pixel and a resolution of 1360 x 1024 x 8 pixel. Exposure times were maintained fixed while reading the CYP1A1, CYP2B and MICA for each treatment. A series of images of each slide was acquired so that a minimum of 250 cells/slide could be counted. The total fluorescence revealed by the program is divided by number of cells to obtain arbitrary unity of fluorescence (AUF) per cell. Several slides for CYP1A1, CYP2B and MICA were made for each culture: one was a blank (cells treated only with primary and secondary antibodies), one was a secondary blank (cells treated only with secondary antibody), one was a chemical blank (cells treated with contaminant carrier), two were for each treatment dose of contaminants. The blank enabled the natural presence of the target proteins in cultured fibroblasts to be checked. The secondary blank enabled validation of the dose of secondary

Fibroblast cell cultures (third generation) of striped dolphin (RT23) were subjected to this experimental protocol for 4 h. A cell line was exposed to a mixture of benzo(a)pyrene

**OC mixture** 

**PAHs** 

**Flame retardants** 

**BPA** 

(Arochlor 1260 + pp'DDT + p'DDE) solubilized in DMSO (0.05%)

Benzo(a)pyrene (1mM) + betanaphthoflavone (20mM) solubilized in acetone (0.1%)

BDE-MXE solubilized in nonane (0.01 μg/ml)

BPA solubilized in ethanol (0.1%)
